Wastewater Proficiency Testing (PT WWS4) Report
Summary of Key Findings
- A total of 22 laboratories participated in this exercise
- Assay performance was clearly genotype-dependent; however, interpretation should be limited to the specific measles spike materials used for this exercise
- The Paulos et al. assay (WWSCAN) showed the highest sensitivity for the D8 spike, but failed to detect the B3 spike (Figure 1)
- False detections (cross-reactivity) were rare, though some misclassifications of the B3 genotype and vaccine-derived signal did occur (most commonly with the Wu et al. assay implemented with a 55 degree C annealing temperature, Figure 3)
- Whole genome sequencing data were critical to understanding assay performance Section 2.2
- Unexpected assay behavior was observed during internal testing, with likely artifactual amplification observed with a clinical assay, specific to the D8 genotype (Section 4.2)
Cross-Assay Comparisons
Participating laboratories tested PT samples using the following assays:
- Hummel et al. (2006): A clinical assay that does not discriminate between wild-type and vaccine-derived signal. No participating public health laboratories are using this assay for their routine measles surveillance; the datasets represented here come from WSLH internal testing and a participating commercial laboratory.
- Wu et al. (2024): A widely-used assay developed by the Stadler Lab at Rice University. Implementation of this assay varies widely by laboratory, both in what probes are used and the thermal cycling conditions used. The conditions used for internal testing are aligned with recently-released CDC recommendations (62 degree annealing temp and WT1 probe alone).
- Paulos et al. (2025): A modified version of an assay published in Roy et al. (2017), employed by WastewaterSCAN as well as other programs for routine surveillance.
- GT Molecular: Target sequences for commercial assays #100835 and #100831 are not publicly disclosed. GT kit #101035 represents a commercial implementation of a recently CDC-recommended assay for wild-type measles and corresponds to the Wu et al. assay, employing the WT1 probe only. Results from catalogue #101035 are therefore grouped with Wu et al. for the purposes of this analysis.
| Assay Name | # of Results |
|---|---|
| Wu et al. (Rice) | 10 |
| GT Molecular | 7 |
| Paulos et al. (WWSCAN) | 6 |
| Hummel et al. | 2 |
Assay Regions
The diagram below illustrates the regions of the measles virus targeted by assays used by participating laboratories, as well as the N450 genotyping region.
Sequence Information
The following plots display the primer and probe sequences of the PCR assays used by participating laboratories alongside reference material sequences for the corresponding assay regions. Positions displayed in red text indicate a mismatch between primer or probe sequences and sequenced reference material; black text indicates the presence of a degenerate base in the primer sequence.
Wu et al. Assay (Rice University, Stadler Lab)
Paulos et al. Assay (WWSCAN, modified Roy et al.)
Hummel et al. Assay (CDC clinical assay)
Assay x Genotype Effects
Interpretation of assay performance across genotypes is limited to the spiked reference materials used in this PT only.
The assays used showed quite different sensitivity to the B3 and D8 genotypes. The Paulos et al. assay provided the highest quantification for the D8 spike, while the Hummel et al. assay exhibited the highest sensitivity for the B3 spike. The Paulos et al. assay failed to detect the B3 spike used in this exercise. This is likely attributable to the single base mismatch to the B3 template with Paulos et al. probe (note that this wild-type specific assay also harbors only a single mismatch to the vaccine genotype used here). However, we should stress that internal validation of the Paulos et al. assay was successfully completed using clinical RNA of the B3 genotype (from an alternative source, with sequence unknown). This further highlights the importance of sequencing data for interpreting assay performance.
No participating laboratories reported the presence of wild-type measles (above LOD) in the ‘WW Only’ background sample, and only one laboratory reported a detection of wild-type signal in samples containing only the vaccine genotype. Of the assays represented, erroneous trace detections were the most common in the Paulos et al. assay.
Assay Condition Effects
Implementations of the Wu et al. assay as well as the Paulos et al. assay vary across laboratories, particularly with respect to PCR annealing times and temperatures. Statistical power is limited by small sample sizes, but a few key conditions are plotted in more detail below.
Higher annealing temperatures (62 degrees C) employed with the Wu et al. assay generally resulted in lower quantification of both B3 and D8 genotypes. Multiple labs that used a 55 degree annealing temperature mis-classified the B3 genotype as vaccine-derived signal. Note that only a small number of participating laboratories (n = 4) employ a vaccine-specific assay.